Protein translocation and biogenesis machines Abstract Cells are compartmentalized by membranes and macromolecules must cross these barriers. The SecY/Sec61 complex and the Nuclear Pore Complex (NPC) mediate transport across single and double membranes, respectively. Secretory and membrane proteins are translocated by the bacterial ribosome- SecY complex and by the 80S-Sec61 complex. The NPC is a gateway for proteins and RNPs to cross the nuclear envelope and is comprised of a spoke ring complex and a central transporter. Aim 1- We will elucidate the mechanism of co-translational translocation by determining structures of ribosome-SecYEG complexes with a defined nascent chain, to give novel views of early translocation intermediates. To this end, stalled translocation intermediates are trapped by cross-linking in E. coli, purified and reconstituted in nanodisks with native E. coli lipids. Frozen-hydrated particles are then imaged with a Volta Phase Plate to give informative maps after multi-body refinement. Active complexes will be compared to the ground state channel to provide a detailed view of this stepwise process. This work may also provide insights into the positive inside rule for membrane protein topology. An N-terminal, 2-helix bundle in SecE of medically-relevant enterobacteria, such as E. coli (O157:H7), may provide a novel antibiotic target, once we understand its role in translocation. Our pipeline will provide a path forward to study Signal Sequence Peptidase interactions with SecYEG, membrane protein integration and the role of non-canonical signal sequences in translocation. Finally, a structure of the TRAP complex will help us to understand how nascent chains with weaker signal sequences may be translocated more efficiently by the ribosome-Sec61 complex. Aim 2- We recently determined a first-pass, domain structure of the yeast NPC, which revealed the functional anatomy of this translocation machine. A composite 3D map will now be determined at higher resolution with single particle cryo-EM and/or state-of-the-art tomographic sub-volume averaging. The new 3D map will be combined with a large database of nucleoporin (Nup) crosslinks, ~25 published Nup domain structures, and a structure of the Nup84 complex, to create a multi-scale model of the NPC with Integrative Modeling. This in turn, will provide insights into Nup roles in assembly, pore stabilization, transport, and evolution of the NPC. Three-dimensional classification will reveal the density distribution of FG Nups in the central transporter, which will guide Brownian Dynamics simulations to dissect the transport mechanism.